Background: The combination of the CFTR corrector lumacaftor (LUM) and potentiator ivacaftor (IVA) has been labeled in France since 2015 for F508del homozygote cystic fibrosis (CF) patients over 12 years. In this real-life study, we aimed (i) to compare the changes in lung function, clinical  and radiological parameters, and in sweat chloride concentration before and after initiation of LUM/IVA treatment; (ii) to identify factors associated with response to treatment; and (iii) to assess the tolerance to treatment.Materials and Methods: In this tri-center, non-interventional, and observational cohort study, children (12–18 years old) were assessed prospectively during the 2 years of therapy, and retrospectively during the 2 years preceding treatment. Data collected and analyzed for the study were exclusively extracted from the medical electronic system records of the patients.Results: Forty adolescents aged 12.0–17.4 years at LUM/IVA initiation were included. The lung function decreased significantly during and prior to treatment and increased after LUM/IVA initiation, becoming significant after 2 years of treatment. LUM/IVA significantly improved the BMI Z-score and sweat chloride concentration. By contrast, there was no significant change in exacerbation rates, antibiotic use, or CT scan scores. Age at LUM/IVA initiation was lower in good responders and associated with greater ppFEV1 change during the 2 years of treatment. LUM/IVA was well-tolerated.Conclusion: In F508del homozygote adolescents, real-life long-term LUM/IVA improved the ppFEV1 trajectory, particularly in the youngest patients, nutritional status, and sweat chloride concentration but not exacerbation rates or radiological scores. LUM/IVA was generally well-tolerated and safe. CFTR is involved in active excretion of chloride at the apical membrane of the epithelial cells (Cystic fibrosis (CF) is an inherited genetic disease leading to al cells –4. Amongal cells . This mual cells , 4, 6. Aal cells  and orgaal cells –10. Theral cells , 12 slowal cells , 10. Theal cells .In this context, therapeutic approaches have been developed to improved cell chloride secretion by targeting the defect in F508del-CFTR folding using in association: the CFTR corrector lumacaftor (LUM) (enhances trafficking and processing), and the CFTR potentiator ivacaftor (IVA) (increases the channel opening probability) , 4. PhasThus, real-life studies are needed to confirm the ability of LUM/IVA to change the course of the disease and to evaluate its long-term effects, particularly in adolescents, who are at a critical stage of their development.Z-scores) and number of pulmonary exacerbation, antibiotic use], biological , and radiological  mucus secretions sub-score) parameters; (ii) to identify parameters associated with response to treatment; and (iii) to assess the tolerance to treatment.Thus, we conducted a pediatric prospective real-life cohort study including F508del homozygote adolescents followed-up within the French South-Western network (MUCOSUD) to assess the evolution of the ppFEV1 before and after the LUM/IVA initiation. The secondary objectives were to assess (i) the evolution of clinical . Categorical variables were expressed as absolute values and as percentages. Categorial variables were performed using the Fisher's exact test or Chi square test. Correlations were performed using the Spearman test. A p-value < 0.05 was considered significant.Analyses were performed only on available data using R software® (version 4.0). Figures were done using Prism software® (version 5.1). Data from the entire population were analyzed and comparison between subgroups according to the presence or absence of impaired lung function (ppFEV1 < 80%) or according to the Among the 350 adolescents with CF followed within the French South-West network of pediatric CF centers (MUCOSUD), 50 were eligible for the LUM/IVA treatment of whom one had a contraindication for LUM/IVA (severe cirrhosis) and seven were included in new therapies clinical trials . LUM/IVAr = −0.46; p = 0.009) and BMI Z-scores , and a positive correlation between ppFEV1 and BMI Z-score . Except for lower ppFEV1 and BMI Z-scores in patients with impaired lung function (ppFEV1 < 80%) at M0, there was no significant difference between patients according to the presence or the absence of impaired lung function at M0 (Z-scores) and sweat chloride, there was no difference between patients with or without BMI Z-scores < 0 at M0 (The main characteristics of the 40 adolescents at initiation of LUM/IVA are summarized in on at M0 . Except  0 at M0 .p = 0.008) (p = 0.027) (Z-score was stable from M-24 to M0 (p = 0.28), increased after the LUM/IVA initiation with a significant improvement to 0.02 ± 0.92 at M24 corresponding to an increase of 0.31 ± 0.66 compared with M0 (p = 0.006) (Z-score (Z-score (p = 0.002) but remained higher than the upper limit of the normal (From 2 years before (M-24) to LUM/IVA initiation (M0), the ppFEV1 decreased significantly from 88.3% ± 17.0 to 83.4% ± 16.5 (= 0.008) . By cont= 0.027) . To note= 0.027) . In addi= 0.006) . Interes. Prior to treatment, the BMI Z-score remained stable in the two groups  at the LUM/IVA initiation, the ppFEV1 increased significantly from M0 to M24 (ppFEV1 absolute change of +7.3 ± 10.8%) after a significant decrease prior to the LUM/IVA initiation (ppFEV1 absolute change of −9.4 ± 13.3%) . The samo groups . After t4 to M24 . In the 4 to M24 .Z-score < 0 at the LUM/IVA initiation, the ppFEV1 decreased significantly in the 2 years prior to the LUM/IVA initiation but remained stable in the following 2 years , HAV scores (p = 0.482), and Bhalla scores (p = 0.576). When characteristics assessed at M0 of good responders and the other patients were compared, only the age at the LUM/IVA initiation was significantly different and lower in good responders (p = 0.04) (p = 0.57), BMI Z-scores , and the sweat chloride concentrations  at M0, sweat chloride improvement between M0 and M12 , or BMI Z-score change between M0 and M24 .During the LUM/IVA treatment, the number of low/non-responders decreased, and the number of patients in both the no decline group and the good responders group increased significantly with time . After 2itiation . Due to sponders . Indeed,patients . Surprissponders . Moreove = 0.04) , whereasn = 1), increased anxiety (n = 1), transient diarrhea (n = 3), and increased aspartate and alanine aminotransferase >5 times the upper limit of normal (n = 1). Except for this last patient who temporarily interrupted the treatment, all the others maintained their treatment for at least 2 years independently of the observed response and without any difference between good responders and other patients. No cataract or lens opacities were detected.Side effects observed in the 40 patients included were transient chest tightness (Early cessation of treatment (<3 months) occurred in only two patients, due to psychological depression in one case and social difficulties in the other.Z-score < 0 at the LUM/IVA initiation and a concomitant improvement in BMI Z-score and sweat chloride concentration after the LUM/IVA treatment.This real-life study highlights the long-term effectiveness and safety of LUM/IVA on lung function and nutritional outcomes in CF adolescents in real life. We found a significant improvement in the ppFEV1, from worsening in the 2 years preceding treatment to a significant increase during the 2 years of the LUM/IVA therapy, particularly in patients with ppFEV1 < 80% or BMI Z-score < 0, and at a lesser extent in those with normal ppFEV1 or BMI Z-score > 0 at the start of the treatment . Patients with impaired lung function or BMI Z-score < 0 had also the worst sweat chloride which may be related to a poorer nutritional status or greater CFTR dysfunction. Thus, it was not surprising that restoring CFTR function and/or nutritional status with LUM/IVA has a better effect in these patients. Moreover, we highlighted an inverse correlation between the age at the LUM/IVA initiation and improvement in ppFEV1 after 2 years of treatment. This suggests that early initiation of the treatment at a younger age regardless of pulmonary function or growth parameters may be more beneficial. This was in accordance with the improvement in lung clearance index at 24 weeks of treatment in younger patients (9.1 ± 1.5 years old) with normal lung function as previously described in a 6–11 year phase III study   and PROGII study .Z-score), particularly in adolescents with poor BMI Z-scores at the start of LUM/IVA as demonstrated in younger patients included in the LUM/IVA phase III  , 17 or iWe did not find difference in Bhalla  scores oIn our study, the side effects of the treatment were consistent with those observed in the industry-sponsored studies , 15, 17 Our study had several limitations. First, the number of patients included is low compared with other real-life studies and there is no control-group. However, these patients accounted for 84% of our patients eligible for the LUM/IVA treatment in our centers. Second, spirometric tests performed 2 years and 1 year before and after T0 were selected to match the best ppFEV1 obtained at ±1 month from the chosen time point. We use this method to avoid the transient effect of exacerbation on ppFEV1. Third, pre-treatment data were assessed retrospectively. However, such data included standard items, which were uniformly and prospectively recorded during routine follow-up using the same software in the entire South-West network. This data is also channeled to the national registry, decreasing the risk of bias due to missing data. Fourth, only exacerbations requiring antibiotics have been considered, in contrast to less severe pediatric exacerbations due in most instances to viral infections. However, compared with previous clinical trials we assessed exacerbation requiring IV and/or oral antibiotics and not only those requiring IV courses which represent only a subset of antibiotic-treated exacerbations especially in adolescents. Fifth, the adherence to treatment was assessed at each visit but was based on the reports of children or their parents, and this represents an important limit of real-life studies.Z-score, which have been associated with survival in cystic fibrosis. Adolescents with impaired lung function had a greater treatment benefit on lung function, but early introduction appeared to promote better response to the treatment, with good tolerance. In the era of CFTR modulators, real-life studies help the clinicians understand what type of results are really to be expected according to specific age groups.We confirm that in real life, adolescents given long-term LUM/IVA improve their ppFEV1 and BMI This real-life study showed short- and long-term benefits of lumacaftor/ivacafor association in adolescents with cystic fibrosis, particularly in the youngest ones, by changing trajectories and improving lung function and nutritional parameters.This study focuses on the long-term safety and efficacy of lumacaftor/ivacaftor in children with cystic fibrosis in real-life.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.SB designed and supervised the study, with contributions from AM and MM. SB, AM, LR, FG, CC, RE, JL, MM, GD, MF, and EM included and followed the patients. The statistical analysis was performed by SB, FB, and RE. SB, MM, and AM were major contributors in writing the manuscript and contributed equally to the manuscript. All authors have approved the final manuscript as submitted and agree to be accountable for all aspects of the work.SB, FG, CC, RE, FB, and MF conducted clinical trials with Vertex pharmacological agents, on behalf of the European Cystic Fibrosis Society-Clinical Trials Network (ECFS-CTN) and within the scope of ECFS-CTN activities. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Managing violent behavior is a particularly challenging aspect of hospital psychiatric care. Available pharmacological interventions are often unsatisfactory.Aim: To assess the effectiveness and safety of daytime zopiclone add-on administration in violent and difficult-to-treat psychiatric inpatients.Methods: Chart review of inpatients treated with daytime zopiclone, between 2014 and 2018, with up to 12 weeks follow-up. Effectiveness was retrospectively assessed with the Clinical Global Impression rating scale (CGI) and the frequency and severity of aggressive incidents recorded with the Staff Observation Aggression Scale-Revised (SOAS-R).Results: Forty-five  cases, 18–69 years age range, average (SD) baseline CGI-S score of 5.4 (1.0), and a variety of diagnoses. Sixty-nine percent showed CGI-S improvement of any degree. For patients with at least one aggressive incident within 7 days prior to initiation of zopiclone (N = 22), average (SD) SOAS-R-Severity LOCF to baseline change was −3.5 (2.7) P < 0.0001. Most patients reported no side effects; 24% reported one or more side effects, and 11% discontinued zopiclone due to sedation (4), insomnia (1) or slurred speech (1). No SAEs were recorded. Zopiclone maximum daily dose correlated with CGI-S baseline-to-LOCF change . The ROC AUC of zopiclone maximum daily dose and improvement on CGI-S was 0.84 . The ROC AUC of zopiclone maximum daily dose and SOAS-R-N improvement was 0.80  and maximum Youden's index value was achieved at a dose of >30 mg.Conclusions: Zopiclone doses >30 mg daily achieved the best anti-aggressive effect. Zopiclone is a short-acting hypnotic agent with a pharmacological profile similar to benzodiazepines. Zopiclone pharmacological properties are hypnotic, sedative, anxiolytic, anticonvulsant, and muscle relaxant. These effects are related to a specific positive allosteric modulation (PAM) of central receptors belonging to the GABAA macromolecular complex, modulating the chloride ion channel's opening .Zopiclone is often found in the blood of those with drug abuse, self-harm, overdose, and road accidents. It is overused in the elderly, and many adverse consequences, including injury, have been associated with it . ZopicloNonetheless, in 2014 we made a serendipitous observation of remarkable clinical effects after daytime administration of zopiclone in a treatment-resistant patient with a Major Vascular Neurocognitive Disorder.This patient  showed intermittent brief runs of high amplitude 1–3 Hz waves predominantly over the right side leads and frontally and frequent 4–8 Hz waves over the central and temporal regions. Considering a previous report  General Adult Program. AHE is the largest psychiatric hospital in the Canadian province of Alberta. The general psychiatric adult section of the hospital comprises three secure general adult acute units (84 beds) and one secure psychiatric intensive care unit (16 beds) (PICU). The PICU at AHE is the largest in the province and accepts patients directly from all Emergency Departments and acute general psychiatric units in the Edmonton Zone (~1.4 million population). The PICU also accepts transfers from other adult PICUs, and from forensic wards of patients released from any legal hold still requiring PICU level of care. In addition, the PICU receives patients from other services including intellectual disability, rehabilitation, and old age inpatient programs. Consequently, the PICU at AHE is a tertiary multidisciplinary acute inpatient unit, specialized in the care of highly complex and frequently treatment resistant patients posing a high risk of harm to self or others. Most patients described in this paper were initiated on zopiclone while they received care in the PICU.Using our hospital pharmacy database, we identified 65 inpatients who had been prescribed daytime zopiclone between 2014 and 2018 by 12 different physicians. From this list, we excluded: nine patients who had not been adherent to treatment for at least three continuous days; one patient who was tried on zopiclone to improve essential tremor and not for treatment of psychiatric symptoms; finally, we were unable to include 10 patients because their clinical records could not be made available before a set deadline. Therefore, the clinical records of 45 patients were reviewed to extract a narrative summary of the cases, with attention to any reported side effects. For all patients excluded from this retrospective review, any available information about untoward events was included in our safety analysis. The recorded diagnoses  were those proposed by the admitting physician and were never changed by the raters. Retrospectively we rated mental state changes using the Clinical Global Impression rating scale (CGI) , the alpha value 0.05 was corrected to 0.005, using Bonferroni's method. A receiver operating characteristic curve (ROC) was used to determine the optimal dose of zopiclone to improve the outcome measures. ROC is a statistical tool to assess the accuracy of a test. The ROC curve is widely accepted to select an optimal cut-off point and compare diagnostic tests' accuracy. It is a plot of the true-positive rate against the false-positive rate for different cut-off points. Other applications of the ROC curve for various situations in clinical trials and drug development have been proposed .Descriptive statistics were used to quantify demographic and clinical variables. Differences in clinical variable scores before and after treatment with zopiclone were assessed with a non-parametric Mann-Whitney test. The sample was stratified according to different criteria, and efficacy was compared among these groups. When the stratification criterion was dichotomous, the resulting groups could be compared with the Mann-Whitney test. When the stratification criterion was a continuous variable, the association between this criterion and the outcome variable was quantified with Spearman's coefficient of rank correlation (rho). Because of multiple comparisons tested in this study  baseline CGI-S scores were 6 and 5.4 (1.0), where a CGI-S score of 6 indicates “severely ill,” and 5 means “markedly ill.” Median, average (SD) and range of current hospital length of stay (LOS) were 147, 256.3 (322.9), 13–1,435 days. The average (SD) duration of illness was 21.1 (20.5) years.Patients were treated a minimum of 3 consecutive days to a maximum of 498 days, with an average (SD) of 84.4 (113.7) days. The number of zopiclone administrations varied from twice daily to four times daily, with maximum daily dose ranging between 5 and 60 mg, with an average (SD) of 38.0 (18.9).P < 0.0001).Average (SD) CGI-S last observation carried forward (LOCF) was 4.0 (1.3), with 31 (69%) of patients showing a reduction of at least 1 point on the CGI-S and 19 (42%) showing a decrease of at least 2 points on the CGI-S. The average (SD) CGI-S change from baseline to LOCF was −1.5 (1.4) (Average (SD) CGI-I LOCF was 2.5 (1.3), and the number of patients with CGI-I <3 (much or very much improved) were 27 (60%).Average (SD) CGI-EI LOCF was 6.2 (4.7), where 6 stands for “decided improvement with side effects that do not significantly interfere with patient's functioning.”Baseline average (SD) weekly number of aggression incidents (SOAS-R-N) changed from 0.6 (0.8) to 0.2 (0.3)  or 0.1 (0.2) (LOCF), after zopiclone administration. Both differences of SOAS-N from baseline were statistically significant . SOAS-R-Baseline average (SD) severity of aggression incidents (SOAS-R-S) changed from 2.2 (2.4) to 0.7 (0.9)  or 0.3 (1.0) (LOCF) after zopiclone administration. Both differences of SOAS-R-S from baseline were statistically significant .None of the patients, who received zopiclone for more than 12 weeks while still in hospital, showed a marked exacerbation of symptoms after the 12th week of observation.No serious adverse events were reported in any of the 45 patients described here, as well as in the other 20 patients known to have been treated with daytime zopiclone. 11 patients (24%) reported one or more side effects; of these, 5 (11%) discontinued zopiclone. The reported side effects were drowsiness and/or sedation (8), tiredness (3), insomnia (2), slurred speech (1). Side effects leading to discontinuation of zopiclone were drowsiness or sedation (4), insomnia (1), slurred speech (1). All reported side effects were temporary or resolved after discontinuation. There was no statistically significant correlation between zopiclone dose and the onset of side effects.The average (SD) CGI-S change from baseline to LOCF was −1.5 (1.4) for males and −1.3 (1.8) for females (NS). The average (SD) number of aggression incidents change from baseline to LOCF was −0.6 (0.9) for males and −0.5 (0.8) for females (NS). The average (SD) severity of aggression incidents from baseline to LOCF was −2.3 (2.6) for males and −1.6 (2.5) for females (NS).P = 0.046, that is NS after Bonferroni correction).Spearman's rho of age and CGI-S change from baseline to LOCF was 0.1 (NS), rho of age and SOAS-R-N change was 0.2 (NS), rho of age and SOAS-R-N change was 0.2 (NS), rho of age and SOAS-R-S change was 0.3 (The average (SD) CGI-S change from baseline to LOCF in the different diagnostic subgroups were −1.3 (1.6) in Schizophrenia spectrum disorders, −1.9 (1.3) in neurodevelopmental disorders, −1.7 (1.3) in neurocognitive disorders, −1.7 (2.1) in bipolar disorders. None of the group differences were statistically significant. The differences in SOAS-R score change between diagnostic subgroups were also not statistically significant.P = 0.0392, NS after Bonferroni correction)  .P = 0.0044, significant after Bonferroni correction).Current hospital length of stay (LOS) was associated with a lower response to zopiclone, as shown by Spearman's rho of LOS and CGI-S change from baseline to LOCF equal to 0.4 .In patients with aggressive behavior in the 7 days prior to zopiclone initiation, a reduction of SOAS-R-N > 0 was observed in 6/6 patients with excess slow rhythms EEG and 9/11 patients with normal EEG .In patients with baseline aggressive behavior, a reduction of SOAS-R-S > 3 was observed in 4/6 patients with excess slow rhythms EEG and 4/11 patients with normal EEG .An improvement in CGI-S was observed in 17/22 patients with baseline SOAS-R-N > 1 and 14/23 patients with SOAS-R-N = 0 .Zopiclone treatment duration did not predict response. Spearman's coefficient of rank correlation (rho) of zopiclone trial duration and CGI-S, SOAS-R-N and SOAS-R-S change from baseline to LOCF were equal to −0.2, −0.3, and −0.2 respectively, .P = 0.0003, significant after Bonferroni correction).As shown in P < 0.0001). A dose of >22.5 mg achieved Youden's index's maximum value, 84% sensitivity and 71% specificity. Doses >30 mg achieved +LR > 4.P = 0.0017, significant after Bonferroni correction).Rho of zopiclone maximum daily dose and SOAS-R-N score change (LOCF—baseline) was −0.4 .Rho of zopiclone maximum daily dose and SOAS-R-S change from average pre-Z to average post-Z was −0.4 (P = 0.0008) and maximum value of Youden's index achieved at a total daily dose of >30 mg.The ROC analysis using SOAS-R-N improvement as classificatory variable provided similar results, with AUC = 0.80  and maximum value of Youden's index for the ROC curve achieved at a total daily dose of >30 mg.The ROC analysis using SOAS-R-S improvement as a classificatory variable could not be performed in patients with baseline aggressive behavior because all patients had a reduction of SOAS-R-S score irrespective of zopiclone dose. The ROC analysis using SOAS-R-S improvement in all 45 patients, with or without baseline aggressive behavior, resulted in AUC = 0.67  and a median LOS of 147 days, which is markedly higher than the median LOS of 20 days recorded in Canada  and otheFurthermore, there is support for an association between reduced GABA activity and aggression . ConsistIn our case-series an improvement of illness severity (CGI-S) was observed in 69% of patients, with 60% being much or very much improved. Clinical improvement was observed across different mental state dimensions, including cognition in patients with neurocognitive disorders, motricity, mood, and catatonic features. No effect on positive psychotic symptoms was reported in any of the patients. The dimension most often showing improvement, across this diagnostically mixed group of patients, was aggression. While on daytime zopiclone, 91% of patients with baseline aggression had a clinically relevant reduction in aggressive incidents. Treatment was well-tolerated by most patients, with no severe adverse events. The most common side effects reported in our cohort  are also the most reported side effects of zopiclone's nighttime administration. Insomnia was reported in 2 cases, and this paradoxical side effect has also been rarely observed with nighttime administration . Six patThe selection of patients of this study represents both a strength and a limitation. Studies of new treatments for extremely complex and difficult to treat psychiatric inpatients are rare. At the same time, this study provides no evidence to justify the off-label use of zopiclone in the vast majority of patients admitted to psychiatric wards.Another important limitation is the design of this study which is not a prospective randomized double-blind comparator-controlled study. However, this study was not hypothesis driven and it was rather inspired by the desire to better understand a new practice in our hospital, which was started after a serendipitous observation.The inclusion of patients at the peak of severity of their symptoms represents another potential limitation of this study. This is in relation to the spontaneous tendency for patients to improve when they have a maximum of symptoms. This confounder is difficult to quantify in studies without an alternative treatment comparison. However, the correlation between follow-up duration and clinical outcomes was low and not statistically significant (even without Bonferroni correction); which suggests that this phenomenon of spontaneous improvement did not occur in this cohort of difficult to treat patients.P-value). Nevertheless, using the Benjamini-Hochberg method the statistical significance of the 2 variables with uncorrected P < 0.05 remained not significant after correction (adjusted P = 0.1).In statistics, the Bonferroni correction of P is a method to counteract the problem of multiple comparisons which increase the chance of observing a rare event, and therefore, the likelihood of incorrectly rejecting a null hypothesis . We opted for the most conservative method in analyzing the statistical significance of the relationship of multiple demographic and clinical variables to outcomes because of the novelty of our observations and hence the concern of creating false expectations based on a fluke. However, the Bonferroni correction comes at the cost of increasing the probability of producing false negatives, i.e., reducing statistical power. For that reason, new methods of P correction have been proposed  in reducing aggression and other clinical dimensions in difficult-to-treat neuropsychiatric patients, the question about the possible mechanism of action deserves some preliminary exploration. We speculate here about 2 potential competing mechanisms of action. The first mechanism would involve the remediation of thalamocortical dysrhythmia, which has been postulated to be a common pathophysiological mechanism of multiple chronic difficult-to-treat neuropsychiatric disorders .Zopiclone is highly selective for α5 subunit, medium binding to α2, α3 but lower toward α1 on GABAA receptor . It has An alternative mechanism of action of zopiclone would involve immunomodulatory and anti-inflammatory properties.Since the early descriptions of anti-NMDA-receptor encephalitis –27, the The potential therapeutic role of zopiclone in autoimmune psychiatric syndromes could be explained by a non-specific immunomodulatory effect or a specific protection against the neurotoxic effects of autoantibodies for GABAA receptors. In recent years it has been discovered that GABA can modulate the immune response –45. ConsThe original contributions presented in the study are included in the article/This retrospective study, involving human participants, was reviewed and approved by University of Alberta Research Information Services (Pro00072813). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.AC-N and EB: had full access to all of the data in the study and take responsibility for the integrity and the accuracy of the data analysis. AC-N: concept and design and statistical analysis. AC-N, DL, LB, and EB: drafting of the manuscript. AC-N, EB, DL, LB, ZL, and MS: critical revision of the manuscript for important intellectual content. All authors acquisition, analysis, or interpretation of data.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


A contrast-enhanced computed tomography (CT) scan of the abdomen revealed a large gas-forming abscess measuring 12 cm×11 cm×9 cm in the right lobe of the liver (Klebsiella pneumoniae was cultured from both blood and pus. With a gas-forming pyogenic liver abscess (GFPLA) diagnosis, he received six weeks of antibiotic therapy and had a satisfactory response to the medical treatment without any complications.A 57-year-old man with poorly controlled diabetes mellitus (DM) was admitted to our hospital with a four-day history of fever, chills, and mild right upper quadrant pain. On physical examination, mild tenderness in the right upper quadrant of the abdomen was observed. Laboratory data showed leukocytosis (15.3×10he liver . The patK. pneumoniae is the most common pathogen of GFPLA and is found in 85.9% of positive liver pus cultures in GFPLA patientsK. pneumoniae infection in DM patients is the cornerstone for the development of GFPLA,GFPLA is an uncommon and potentially fatal disease, accounting for 5.6-31.8% of pyogenic liver abscesses


In this work, we investigate the TD-DFT simulation, optical, and mirrorless laser properties of conjugated polymer (CP) Poly[-co-], also known as (PFO-co-PPV-MEHB) or ADS125GE. TD-DFT calculations were performed for three monomer units with truncated tails using time-dependent density functional theory (TD-DFT) calculations. The calculations showed a highest occupied and lowest unoccupied molecular orbital (HOMO-LUMO) structure and a very high oscillator strength of 6.434 for the singlet-singlet transition at 374.43 nm. Experimentally, the absorption and fluorescence spectra were examined at various concentrations in verity of solvents, such as benzene, toluene, and hexane. The experimental results obtained in hexane were comparable with theoretical UV-VIS spectra calculated under vacuum. Amplified spontaneous emission (ASE) spectra peaked at approximately 509 nm for CO PFO-co-PPV-MEHB in solution and were obtained at suitable concentrations and pump energies. Additionally, the photochemical stability of this CP and coumarin (C510) were compared. Time-resolved spectroscopy (TRS) studies with a sub-nanosecond resolution were performed for the CO under various pump energies. These results showed the excited state dynamics and single-pass optical gain of CO PFO-co-PPV-MEHB. Since their discovery ,2, four Many CPs exhibit amplified spontaneous emission (ASE), mirrorless lasing with high intensity, spatial coherence, and low temporal coherence. CPs as active gain media have produced ASE and lasing, which could be tunable over a wide range in the visible spectrum ,19,20. BThe first report on ASE from CPs was presented by Moses et al. MEH-PPV produced ASE via optical pumping in solution and film. The quantum efficiency of MEH-PPV was compared to that of the conventional dye rhodamine 6G . AnotherIn 2007, Redmond’s team studied the first incidence of single-nanowire lasing under optical pumping for CO PFO, in which nanowires were formed through the template wetting method and exhibited a single Fabry–Pérot mode . The thrCopolymerization is a technique that enables existing monomers to be combined to achieve new bandgaps and emission properties. The ASE properties of copolymer PFO-co-pX were studied by S.A Alfahd et al. There have only been minimal studies carried out on copolymers with a combination of PFO and PPV derivatives.HOMO, ELUMO, EGap; the dipole moment; and the oscillator strength of PFO-co-PPV-MEHB for structures optimized under the three-monomer truncated-tail oligomer model using the DFT/Coulomb-attenuated method at the B3LYP (CAM-B3LYP)/6-31G level by Gaussian 16 and other software. Furthermore, experimental studies of PFO-co-PPV-MEHB spectral and mirrorless laser properties in toluene under transverse excitation were performed. The pump source was the Nd: YAG laser of 355 nm. We demonstrate that under a suitable concentration and a low pump energy, CO PFO-co-PPV-MEHB can produce ASE at 508 in toluene. TRS studies display the ASE in 3D features, with the wavelength, spectral amplitude, and time as the X-, Y-, and Z-axes, correspondingly.However, in general, DFT simulation, optical, and laser studies of conjugated polymers are rare ,29,30,31The molecular structure of CP PFO-co-PPV-MEHB could be explained as a group of bridged structures. The molecule (A) 1-methyl-4-[(1E)-prop-1-en-1-yl] benzene shown in Seg. A is connected to both sides of the familiar CP poly (PFO) shown in Seg. B. Molecular segment A is connected to another polymer  resembling the MEH-PPV structure, as shown in Seg. C. The cumulative structure becomes [(A)-(B)-(A)-(C)] n. This approach is taken to modify the HOMO-LUMO gap. The calculated dipole moment is 1.4334525 Debye for n = 3, which is for the structure optimized using the DFT/Coulomb-attenuating method (Becke-3 Parameter-Lee-Yang-Parr) CAM-B3LYP/6-31G basis set and the augmented structure is shown in −1 as its molecular weight (Mw). American Dye Source, Inc.  provided the CP material. (ADS125GE) The Coumarin 510 has an Mw of 318.4 g/mol and was precured from Exciton Luxottica. A Perkin Elmer Lambda 950 spectrophotometer  was utilized to record the absorption spectra. The fluorescence spectra at an excitation wavelength of 355 nm from a xenon flash lamp were investigated utilizing a spectrofluorometer . To achieve transverse pumping, the tripled frequency (355 nm) of a 5 ns Nd:YAG laser was focused by a quartz cylindrical lens . The cylindrical lens focused the pulse onto a horizontal strip of 1 cm × 2 mm (length × width). One fiber was linked to an Ocean Optics spectrograph  to record the spectra. Another fiber was linked to a Princeton Instruments PI-MAX 4 ultrafast-gated emICCD, which had a gate delay of less than 500 ps. The camera had a high-speed electrically controlled shutter that allowed events to be recorded with a sub-nanosecond time resolutions .The monomer of PF-co-MEH-PPV is very large and requires significant computational resources. The full-scale simulation of the complete polymer structure with many monomers could be challenging. However, an oligomer structure with three connected monomers is accepted in practice for simulating polymer properties; hence, we adopted the n = 3 oligomer approach. To further reduce the computational burden, we cut short the tail of PFO and MEH-PPV segments, as shown in CPs consist of a large number of atoms; hence, the simulation of CPs is considered to be challenging. Thus, many studies have adopted an oligomeric model of the compound ,36,37. TThe UV-VIS spectrum for PFO-co-PPV-MEHB (n = 3) in vacuum was simulated using the DFT/CAM-B3LYP/6-31G basis set, as shown in Hexane has a very low dielectric contestant and is a nonpolar solvent; hence, its effect on any molecule (solute) is much less than that of most solvents. Hence, it is optimal for comparison with the results simulated in vacuum. The difference between the simulated and experimental absorption spectra (g (eV) = 2.7667 + 0.3408 × (1/n). When the value of n is large, the second term tends to become negligible. Thus, the calculated bandgap value of the polymer is 2.7667 eV using the extrapolation method. This value is comparable with the experimentally measured bandgap of 2.756 eV.−4–12.17 × 10−4 mg/mL. It was found that there are three diverse features. The first is a peak at approximately 355 nm and the second is the main peak at 425 nm, with the third being a shoulder at 370 nm. The optical density decreased from a high value to a low value as the concentration decreased. The absorption spectral profile remained the same, and the full width at half maximum (FWHM) of the maximum peak at 425 nm decreased with the decreasing PFO-co-PPV-MEHB concentration. The absorption spectra in benzene are very close to those of PFO-co-PPV-MEHB in toluene. −4 mg/mL. At low concentrations, fluorescence peaks occur at 485 nm and 510 nm, with a tail at 550 nm. Up to a concentration of 0.078 mg/mL, the fluorescence increases; after that, when the concentration (0.156 mg/mL) is further increased, the fluorescence intensity redshifts and the spectral profile shifts toward red. At higher concentrations, the primary peak of the fluorescence spectrum becomes a shoulder and the 515.5 nm peak becomes dominant with reduced intensity. This behavior is common to fluorescent organic molecules. At low concentrations, the fluorescence output is low because the number of molecules is low in a unit area of solution. As the concentration increases, the intensity also increases up to an optimal concentration. Beyond this concentration, the fluorescence output intensity starts decreasing due to the proximity of molecules, which suppress the certain singlet vibration and increase the reabsorption. The solvent effect was studied in benzene and the trends of the fluorescence intensity and spectral profile were very similar, with a 4 nm redshift in the peak wavelengths at approximately 483 and 508 nm, as displayed in Amplified spontaneous emission occurs due to the stimulated emission and amplification of spontaneously emitted photons under a high population inversion with the single-pass gain of the laser media. ASE without feedback can be considered mirrorless lasing, since most of the features are laser features . Many CP2), the laser-induced fluorescence (LIF) was verified and had two peaks at 507 and 481 nm, as shown in 2, the spectrum became narrower and peaked at 508 nm with an FWHM of 20 nm. When the pump energy density was 7.75 mJ/cm2, ASE was obtained with an FWHM of 8.75 nm and a peak at approximately 508 nm, as presented in PFO-co-PPV-MEHB in toluene at a concentration of 2.5 mg/mL was transversely excited with a 355 nm Nd: YAG laser. At a minimal pump energy density  of ASE was sustained. The reduction in bandwidth (nm) occurred due the net gain maximization near the vibronic transition peaks of the fluorescence spectrum; so, the spectrum displayed intensity surge as the pump energy density raised.2. The C510 shows signs of degradation, even though the output was high. On the other hand, both the stability and high output of CP did not change much; this shows that CP is a very stable laser material. The CP output was not significantly reduced even after twenty thousand (20 × 103) shots. The CP output was almost maintained, as presented in Commonly, photostability is estimated by recording the total ASE intensity emitted under a constant pump energy as a function number of pulses or time. The photodegradation is predictable when a decline in the total ASE output intensity is detected . The ASE2, as shown in 2. The CP started fluorescing at 18 ns and maintained this fluorescence until 69.8 ns. The LIF had two peaks at 507 and 481 nm, and the intensity was unstable, fluctuated unsteadily, and reached a maximum value at 48.19 ns.We began the TRS studies with a PFO-co-PPV-MEHB solution at a concentration of 2.5 mg/mL and a pump energy density of 850 µJ/cm2. The CP started fluorescing at 24 ns, and the intensity increased with time. The CP produced ASE at 22 ns and reached a maximum intensity at 25 ns. After 25 ns, the ASE intensity decayed up to 34.5 ns and stopped at 35 ns, as displayed in The ASE of the CP became intense for pump energy densities above the threshold and was recorded using a picomax spectrometer. 2. When the pump energy density was 7.75 mJ/cm2, ASE was obtained at 520 nm with an FWHM of 8 nm. The entire ASE event became short due to rapid excitation, population inversion, and stimulated emission. The ASE attained the peak in a rapid phase and lasted for only 4 ns. In the pump pulse tailing phase, the photon flux was lower and produced fluorescence at the vibrational band around 480 nm, producing LIF along with weak ASE 2 ns after the maximum ASE peak intensity.This report investigated the theoretical, optical, and mirrorless laser properties of CP PFO-co-PPV-MEHB. The calculation showed a HOMO-LUMO structure and a very high oscillator strength of 6.434 (a.u.) for the singlet-singlet transition at 374.43 nm. The experimental UV-Vis spectra obtained for the nonpolar solvent hexane were analogous to the simulated HOMO–LUMO structure. The simulation showed three oscillator strengths that matched the two peaks and shoulder manifested in experimental UV-VIS spectra, indicating that highly efficient fluorescence properties arise from singlet vibrational transitions. Under an optimal solution concentration and pump energy, CP spectral narrowing was achieved at 508 nm with an FWHM of 8.75 nm. The photochemical stability under pulsed laser excitation was excellent, without deuteration for up to 20 k pulses. TRS studies showed a rapid intensity increase and spectral narrowing within 2 ns of fluorescence.


West Nile virus (WNV), a member of the Japanese encephalitis virus (JEV) serocomplex group, causes lethal encephalitis in humans and horses. Because serodiagnosis of WNV and JEV is hampered by cross-reactivity, the development of a simple, secure, and WNV-specific serodiagnostic system is required. The coexpression of prM protein and E protein leads to the secretion of subviral particles (SPs). Deletion of the C-terminal region of E protein is reported to affect the production of SPs by some flaviviruses. However, the influence of such a deletion on the properties and antigenicity of WNV E protein is unclear. We analyzed the properties of full-length E protein and E proteins lacking the C-terminal region as novel serodiagnostics for WNV infection. Deletion of the C-terminal region of E protein suppressed the formation of SPs but did not affect the production of E protein. The sensitivity of an enzyme-linked immunosorbent assay (ELISA) using the full-length E protein was higher than that using the truncated E proteins. Furthermore, in the ELISA using full-length E protein, there was little cross-reactivity with anti-JEV antibodies, and the sensitivity was similar to that of the neutralization test. Flavivirus in Family Flaviviridae and is a member of the Japanese encephalitis serocomplex group, which includes Japanese encephalitis virus (JEV), Murray Valley encephalitis virus, and Saint Louis encephalitis virus1. In nature, WNV is maintained in a cycle between vector mosquitos and reservoir birds and can cause lethal encephalitis in mammals, including humans and horses2. Before the early 1990s, WNV was limited to Africa, the Middle East, West Asia, and Europe3. In 1999, WNV was reported for the first time in New York City and spread rapidly across North and South America4. In Russia, since a human outbreak of WNV infection was reported in the Volgograd Region in 1999, the endemic area has spread nationwide, including the Far Eastern region7. Because of its wide distribution, a system that enables the detection of WNV in birds and mammals is required to facilitate epidemiological investigations8.West Nile virus (WNV) belongs to the genus 9. Because WNV and JEV frequently exhibit cross-reactivity in serodiagnosis, the distinction of these viruses is important in areas endemic for WNV or JEV10. There are several serodiagnostic tests for WNV infections, such as the enzyme-linked immunosorbent assay (ELISA), neutralization test (NT), immunofluorescence assay (IFA), and hemagglutination-inhibition test. The NT is the most reliable and specific method for the serodiagnosis of flavivirus infection11. However, the NT is time-consuming and requires a Biosafety Level 3 facility to handle live WNV11. By contrast, ELISA is simple and safe, but it is hampered by cross-reactivity among flaviviruses13. Therefore, development of a simple and secure detection system that can distinguish between WNV and JEV is necessary for the serodiagnosis of WNV infection.JEV, which also causes encephalitis, is currently endemic in East Asia14. The viral genome is packaged with multiple copies of capsid protein within a host-derived lipid bilayer, surrounded by both envelope (E) and membrane (prM/M) proteins16. E proteins are located at the surfaces of virions as homodimers in parallel and mediate binding to the host receptors and viral membrane fusion during entry17. The E protein is also the major antigenic site for neutralizing antibodies18. The E protein consists of three ectodomains (Domain I–III) and a C-terminal region including two α-helical regions (H1 and H2) in the stem and transmembrane anchor regions (TM1 and TM2)19. Because the ectodomains have the major antigenic epitopes, E proteins are often used as antigens for serodiagnostics and vaccines22. The stem-anchor region is involved in the membrane incorporation of E protein, stabilization of prM-E interactions, and low-pH-induced membrane fusion associated with conformational changes26. The coexpression of prM and full-length E proteins in mammalian cells results in the production of subviral particles (SPs), which are composed of a viral envelope without capsid protein or viral RNA28. Because the antigenicity of SPs is similar to that of authentic virions, SPs are useful antigens for serological diagnosis30. As the epitope cross-reactive among flaviviruses is less exposed on SPs, an ELISA using SPs enabled the specific serodiagnosis of tick-borne encephalitis virus (TBEV) infection11. By contrast, coexpression of truncated E proteins lacking part or all of the C-terminal region and the prM protein suppressed the formation of SPs24. Truncation of the C-terminal region promoted more efficient secretion of the TBEV E protein compared to the full-length E protein27. However, little is known about the influence of truncation on the secretion and antigenicity of the E protein of WNV.The virions of WNV are approximately 50 nm in diameter and contain a single-strand positive-sense RNA genomeIn this study, we constructed an expression system for full-length E protein and various truncated E proteins lacking the C-terminal regions with One-STrEP-Flag (OSF) tags and analyzed their properties and antigenicity. As a result, we developed a novel serodiagnostic system with high sensitivity and specificity comparable to those of the NT.27. Deletion of the stem-anchor region or insertion of a sequence into the stem-anchor region affected the formation of viral particles, but the E protein was released extracellularly31. To examine the importance of the C-terminal region for the expression and release of E protein, we constructed plasmids expressing prM and various truncated E proteins with an OSF tag at the C terminus to retain the structure of the ectodomain. To expose the OSF tag on the surface of each particle, the sequence was inserted at the N terminus of full-length E protein as reported previously31  and transmembrane regions (TM1 and TM2) termed the stem-anchor region, which connects the E protein ectodomain to the viral membrane and H2 a3, which coincides with that of flavivirus SPs, and the E protein signal was shifted to low-density fractions following treatment with Triton X-10032 . Consistent with the result of ∆C47 with the OSF tag at the C-terminus of E protein (∆C47 C-tag), the ELISA signal of ∆C47 N-tag was significantly lower than that of full-length E protein. This result suggested that the ∆C47 could not form SPs and that the location of the OSF tag unaffected the ELISA.In serodiagnosis, cross-reactions frequently occur among sera from animals infected with flavivirus, especially following WNV and JEV infection. To examine the specificity of the ELISA, sera from mice infected with WNV, JEV, or TBEV were tested. The neutralizing antibody titer of each serum from mice inoculated with WNV, JEV, or TBEV was examined by neutralizing test. Each serum sample for ELISA was standardized by diluting to the same neutralization titer. The ELISA signal of sera from mice inoculated with WNV was significantly higher than that from mice inoculated with JEV, TBEV, or mock Fig. A. No difTo examine the sensitivity of the ELISA, serum samples from mice infected with WNV were tested using an IFA or NT and the results were compared with those of the ELISA. The ELISA signal was strongly correlated with the serum titers from the IFA Fig. B and NT We analyzed the properties of OSF-tagged full-length WNV E protein and various truncated WNV E proteins lacking the C-terminal region and used them in a serodiagnostic ELISA for WNV infection. The C-terminal region of E protein influences particle formation but not the expression and secretion of E protein. Also, the structure of E protein was important for the sensitivity and specificity of the ELISA.36. Solidification of viral particles directly onto ELISA plates impaired the antigenic structure of the native epitopes of E protein37. Because full-length E protein, which formed SPs, was captured by Strep-Tactin on the plate, our system conserved the native epitopes and detected antibodies in serum with high sensitivity. Furthermore, the amount of full-length E protein precipitated by Strep-Tactin was less than that of truncated E protein, whereas ELISA using the full-length E protein exhibited higher reactivity than that using truncated E protein. These results suggest that the reactivity of ELISA in a single SP is much higher than that of a single monomer or dimer of E protein. Therefore, the structure of E protein was more important than its quantity for the sensitivity of the ELISA.The ELISA using full-length E protein, which formed SPs, exhibited higher reactivity than that using truncated E proteins. Furthermore, disruption of the structure of full-length E protein decreased the reactivity to anti-WNV antibody in the ELISA, suggesting that the structural arrangement of E protein influenced the sensitivity of the ELISA. Several antibodies in flavivirus-infected serum could bind to not only the epitopes of E protein at the intra-dimer but also the epitopes at the inter-dimer1. Many serological tests such as commercial or in-house IgG-ELISAs or IFAs exhibited cross-reactivity among members of the JE serocomplex39. The cross-reactive epitopes are thought to be inaccessible on infectious virions or SPs but may be exposed by conformational changes in E protein during these assays, such as during plate coating or fixation41. In our ELISA, the capture of SPs with an OSF tag by Strep-Tactin might have conserved the native structure of the viral particles and epitopes.Our ELISA using OSF-tagged SPs exhibited little cross-reactivity with anti-JEV antibodies in mouse serum. The cross-reaction are often observed among flaviviruses42. The role of the stem-anchor region in the expression and secretion of E protein may differ among flaviviruses. The transmembrane regions of E protein were required for the production of SPs of TBEV and yellow fever virus43. Our sucrose gradient ultracentrifugation results indicated that WNV E protein lacking both transmembrane regions could not form SPs, suggesting that the transmembrane regions of the WNV E protein are important for particle assembly. Furthermore, the sedimentation of full-length E protein generated two distinct peaks in the SPs fraction. In a previous study examining TBEV, SPs of two distinct sizes were separated by sucrose gradient centrifugation, of which the smaller particles were mature SPs and the larger particles were the immature form of SPs44. SPs produced from the full-length E protein of WNV seem to be include both the mature and immature forms. Investigating the antigenicity of each type of SP may facilitate the development of a detection system.Deletion of the stem and anchor regions of WNV E protein did not affect the expression and secretion of E protein, despite incomplete SPs formation. Deletion of the stem-anchor region of TBEV E protein increased the secretion of E protein, whereas deletion of dengue virus (DENV) E protein did not influence expression or secretion47. WNV is transmitted between avian hosts and mosquito vectors in nature48. Further improvement and application of our ELISA serodiagnostic system to detect avian infection by WNV will facilitate investigations of the epidemiology of WNV in nature.In conclusion, we demonstrated that WNV SPs with an OSF tag can be used in a highly sensitive and specific serodiagnostic system for WNV infection. Given the common properties of the SPs of flaviviruses, the system constructed in this study can be applied to the serodiagnosis of other flaviviruses2 and shaking at 125 rpm.Human embryonic kidney 293 T (293 T) cells were cultured at 37 ℃ in high-glucose Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin. Vero cells and baby hamster kidney fibroblast (BHK cells) were cultured in Eagle’s minimum essential medium (Wako) containing 10% FBS and used for neutralization tests. Expi293F cells were cultured in Expi293 Expression medium  at 37℃ with 8% CO49. The JEV JaGAr-01 strain or the TBEV Oshima 5–10 strain was propagated in BHK cells. All WNV and TBEV experiments were performed at a Biosafety Level-3 facility at Hokkaido University, Japan, according to the institutional guidelines, as described previously50.The WNV 6-LP strain was established from the WNV NY99-6922 strain as described previously5 PFU/0.1 mL of JEV (n = 3), or 104 PFU/0.1 mL of TBEV (n = 3). The mice were housed in a climatically controlled environment (24 ± 2 °C) under a 12 h (7 am/7 pm) light/dark cycle with access to chow and water ad libitum. The mice were euthanized when more than 10% of their body weight was lost, and blood samples were collected from the heart under anesthesia and kept at 4 ℃ overnight. The sera were collected and inactivated for 30 min in a heat block at 56 ℃ and stored at − 80 ℃ until use.Eight-week-old female BALB/c mice were purchased from Japan SLC Inc. . Mice were anesthetized using sevoflurane and inoculated subcutaneously in the back region with 100 plaque forming units (PFU)/0.1 mL of WNV (n = 3) or 10The pCAG-WNV-prME plasmid was constructed by subcloning the PCR-amplified prME coding regions of WNV. The sequence of the OSF tag was amplified using PCR from the pCAG-OSF plasmid, generously provided by Dr. Kamitani , and it was then introduced at the N terminus of the E protein in the pCAG-WNV-prME plasmid using an In-Fusion HD Cloning kit  and named pCAG-WNV-prM-OSF-E.51 using appropriate primers via inverse PCR and named pCXSN-prME∆C47. The OSF tag was inserted at the C terminus of the E protein in pCXSN-prME∆C47 using an In-Fusion HD Cloning kit  and named pCXSN-prME∆C47-OSF. The sequence of prME∆C47-OSF was amplified using PCR from pCXSN-prME∆C47-OSF and cloned into the pCAGGS vector plasmid. The resultant plasmid was named pCAG-prME∆C47-OSF. The pCXSN-prME∆C71-OSF, pCXSN-prME∆C88-OSF, and pCXSN-prME∆C101-OSF plasmids were constructed from pCXSN-prME∆C47-OSF via inverse PCR using appropriate primers. The sequence of the insert of each plasmid was amplified using PCR and cloned into the pCAGGS vector plasmid; the resulting plasmids were named pCAG-prME∆C71-OSF, pCAG-prME∆C88-OSF, and pCAG-prME∆C101-OSF, respectively. The pCAG-prM-OSF-E∆C47 was constructed from pCAG-WNV-prM-OSF-E via inverse PCR using appropriate primers.Forty-seven amino acids were truncated from the C terminus of the E protein in pCXSN-prME, which was constructed previouslyPlasmids were transfected into 293 T or Expi293F cells using X-tremeGENE HP DNA Transfection reagent  or an ExpiFectamin 293 Transfection kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Transfected 293 T and Expi293F cells and culture supernatant were harvested at 48 h post-transfection and stored at − 80℃.51. The supernatant from transfected cells was precipitated as described below.Transfected cells were washed and lysed with lysis buffer . The lysates were prepared as described previously51. Briefly, blocked membranes were incubated overnight with an anti-WNV E mouse antibody . Complexes were detected using horseradish peroxidase (HRP)-conjugated anti-mouse IgG secondary antibody . Protein bands were visualized as described previously51.Samples were electrophoresed in polyacrylamide-sodium dodecyl sulfate gels and transferred to polyvinylidene difluoride membranes. Immunoblotting was performed as described previouslyCulture supernatants of transfected 293 T cells were harvested and treated with 1% Triton X-100. The supernatants were immunoprecipitated or pulled down using an anti-WNV E antibody (Millipore) or Strep-Tactin Sepharose . Immunoprecipitation was performed by incubating the supernatants at 4 ℃ for 3 h with 20 μL of antibody-coupled Surebeads Protein G Magnetic Beads . The supernatants were next incubated with Strep-Tactin Sepharose at 4 ℃ for 2 h. The reaction and elution steps were performed following the manufacturer’s instructions. The precipitated protein was separated using SDS-PAGE and analyzed by immunoblotting as described above.g, at 4 °C for 16 h in a CP80MX centrifuge using the P40ST-1239 rotor . Fractions of 550 μL were harvested from the top layer, and the amount of E protein in each fraction was measured by ELISA.Culture supernatants of transfected 293 T cells were treated with or without 1% Triton X-100 and were analyzed via ultracentrifugation in a 10–50% sucrose gradient in HEPES buffer (50 mM HEPES [pH 7.8] and 140 mM NaCl) at 110,000×Ninety-six-well ELISA plates  were coated with Strep-Tactin AP conjugate , incubated overnight at 4 ℃, and blocked with Block Ace . After washing with PBS containing 0.05% Tween 20, culture supernatants containing the constructed E proteins with an OSF tag as an antigen were added, followed by serum samples (1:200). The antibody for WNV, JEV, or TBEV was detected using Protein A/G conjugated to HRP , with OPD as the substrate. Negative control antigens were prepared from the supernatant of untransfected 293 T or Expi293F cells. The results were calculated as P/N ratios .5 cells/well into Nunclon Delta 96-well plates (Thermo Fisher Scientific) and incubated overnight. Cells in the plates were infected with WNV and incubated at 37 ℃ for 2 days. Infected cells were fixed in 4% paraformaldehyde for 15 min and then washed with PBS. Fixed cells were permeabilized and blocked by incubating in PBS containing 0.1% Triton X-100 and 1% bovine serum albumin (BSA) for 30 min. The cells were incubated with WNV-infected serum diluted in PBS containing 0.1% Triton X-100 and 1% BSA for 1 h. After washing with PBS, cells were incubated with Alexa Fluor 555 goat Anti-Mouse IgG (H + L) Secondary Antibody . All procedures were performed at room temperature. Cells were observed using an IX71N-22FL/PH system , and images were processed using Standard CellSens (Olympus).Vero cells were plated at a density of 1 × 10WNV, JEV, or TBEV particles were incubated with serially diluted sera and inoculated in Vero or BHK cells, respectively. The cells were incubated in a medium containing 1.25% methyl cellulose and 5% FBS for 4 days. Next, the cells were fixed with 10% formalin and stained with 0.1% crystal violet. Serum samples that exhibited an 80% reduction in plaque formation of WNV, JEV, or TBEV were screened. The neutralization titers of mouse sera infected with WNV, JEV, or TBEV were calculated , then each serum was standardized by dilution with PBS to the same neutralization titer.Band intensity was quantified via densitometric measurement of the lanes using Image Lab Software (Bio-Rad) and normalized to the relative density of the loading control in the same blot. The optical density at 450 nm was recorded using a Viento 808 microplate reader .Data were compared among multiple independent groups using one-way ANOVA followed by the Tukey–Kramer or Dunnett test. Statistical significance was established a priori at p < 0.05.Animal experiments were performed according to the basic guidelines for animal experiments of the Ministry of Education, Culture, Sports, Science and Technology of Japan. The experimental protocols were approved by the Animal Care and Use Committee of Hokkaido University  and complied Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.Supplementary Information.


Chenopodium quinoa) and is known as a functional food for its beneficial effects on human health. As the presence of RIPs in edible plants could be potentially risky, here we further characterised biochemically the protein  and explored its possible defensive role against pathogens. Quinoin consists of 254 amino acid residues, without cysteinyl residues. As demonstrated by similarities and homology modeling, quinoin preserves the amino acid residues of the active site  and the RIP-fold characteristic of RIPs. The polypeptide chain of quinoin contains two N-glycosylation sites at Asn115 and Asp231, the second of which appears to be linked to sugars. Moreover, by comparative MALDI-TOF tryptic peptide mapping, two differently glycosylated forms of quinoin, named pre-quinoin-1 and pre-quinoin-2  were characterised. Finally, quinoin possesses: (i) strong antiviral activity, both in vitro and in vivo towards Tobacco Necrosis Virus (TNV); (ii) a growth inhibition effect on the bacterial pathogens of plants; and (iii) a slight antifungal effect against two Cryphonectria parasitica strains.Quinoin is a type 1 ribosome-inactivating protein (RIP) we previously isolated from the seeds of pseudocereal quinoa ( In light of this, the theoretical Mrs of the glycosylated peptides T-6 and T-6′ were 3487.87  and 3635.99, respectively . These predicted possible oligosaccharide structures were retrieved in glycoproteins from plants, such as horseradish peroxidase [Among these, the protein synthesis inhibitor PD-S2-like (A.C.: XP_021750597.1) exhibited a closer identity with the sequences of both the tryptic and chymotryptic peptides derived from purified quinoin. In this framework, we decided to use the amino acid sequence of the protein synthesis inhibitor PD-S2-like (A.C.: XP_021750597.1) as a reference protein. The sequencing of both the obtained tryptic and chymotryptic peptides provided for ~38% of the amino acid sequence of quinoin  and their overlap is reported in ysis see c. Indeedmod tool  allowed roxidase ,26.The strategy allowed the MS mapping of ~99% of the quinoin sequence (251/254 amino acid residues) compared to the protein synthesis inhibitor PD-S2-like (A.C.: XP_021750597.1). Therefore, these data allowed us to confirm that the protein synthesis inhibitor PD-S2-like (A.C.: XP_021750597.1) amino acid sequence is the primary structure of quinoin. In addition, no sequence information was achieved for the N-terminal peptide ‘MQQENKKAWL VLTIAIWVVL QQVNA’  and two A-chain of type 2 RIPs found in Ricinus communis. The multiple alignment analysis showed that, in all the RIPs evaluated, the active site residues  involved in the enzymatic mechanism (Glu177 and Arg180) and in the binding with adenine  were conserved [In order to obtain information regarding the identity/similarity of quinoin with other RIPs and to gain insight into its structural features, we performed a search for sequence homology. Among the numerous sequences showing a degree of identity, ranging from about 50% to about 30%, we focused our interest on those reported in onserved . In partonserved ; (ii) whR. communis belonging to the Euphorbiales order, were used as an outgroup. Two main groups were highlighted by the analysis of the unrooted phylogenetic tree , dianthin-30 (D. caryophyllus) and the saporins , while all other RIPs  were located on a separate branch group (b) all belonging to the Phytolaccaceae family. These findings suggest that quinoin shares common ancestors with lychnin, dianthin-30 and saporins. It is interesting to note from a structural point of view that all the proteins that belonged to group (b) (Phytolaccaceae) had four cysteinyl residues, while quinoin and other members of group (a) were without cysteinyl residues , as shown in P. dioica  using bFinally, in order to carry out a predictive study of the structure of quinoin, we used the online I-TASSER service; the program built a three-dimensional model of quinoin by selecting  the three-dimensional structure of RIPs as a template. For quinoin, the computational modeling calculation generated a single conformer with a C-score of 1.09 indicating the good quality of the predicted three-dimensional structure. The overall fold of the quinoin model, including the secondary structure elements, is shown in From the three-dimensional structural model of quinoin, the position of N-glycosylated Asn231 was also investigated. This asparaginyl residue is part of an evident β-hairpin in the C-terminal region (position 225–237) that, as has been reported by many authors, plays a role in biological activity of RIPs. Indeed, several studies have reported that changes in this C-terminal region can alter: (i) the interaction with membrane models ; (ii) thHura crepitans L. and P. americana when subjected to heat or osmotic stress [P. americana) genome showed that the genes of both the pokeweed antiviral protein (PAP) and the PAP isoforms exhibited a long 5′ UTR containing one intron and cis-regulatory elements associated with diverse biotic and abiotic stresses [The pioneering work of Stirpe et al. reported that the increase in the inhibitory activity of protein synthesis and activity on DNA is due to the presence of RIPs in the leaves of both c stress . Since tc stress ,40. The stresses .C. quinoa, [In this framework, with information on the genome of  quinoa,  and, in  quinoa, .In order to acquire the possible structural differences between quinoin and both pre-quinoin-1 and pre-quinoin-2 in addition to glycosylation, we decided to perform a comparative tryptic peptide mapping, using the quinoin primary structure as a reference peptide profile. Thus, the two proteins were first digested with trypsin and then the mixture of peptides was analysed by MALDI-TOF mass spectrometry.rs of the tryptic peptides retrieved for the digested pre-quinoin-1 and pre-quinoin-2 forms are reported in r of the tryptic peptides obtained from the amino acid sequence of quinoin.The MC. quinoa seeds. Moreover, a thorough analysis of the theoretical glycosylation sites in the quinoin primary structure revealed a second glycosylation site (Asn115 in the NIT consensus sequence), which could justify the presence of these glycoforms. This evidence is in agreement with the different glycosylated glycoforms of PD-S2, the major type 1 RIP characterised in the seeds of P. dioica [Therefore, taking into account the MALDI-TOF-MS peptide mapping, it is important to consider that both pre-quinoin-1 and pre-quinoin-2 type 1 RIPs are different glycosylated forms of quinoin, the major glycoform isolated from . dioica .Several type 1 RIPs are known for their antiviral properties against many virus pathogens for plants, animals and bacteria. In light of this, several authors have proposed these enzymes as possible candidates to combat infectious diseases and, in particular, to control crop virus diseases, also considering the lack of specific antiviral products used in the open field .P. dioica [Thus, we investigated the possible antiviral action of quinoin against TNV, which affects numerous cultivated plants. In a preliminary experiment, quinoin was tested at 2.0 and 10 µg/mL (70 and 350 nM), concentrations that had previously been shown to be effective in the case of PD-L1 and PD-L4, two type 1 RIPs from the leaves of . dioica . At bothP. dioica and Beta vulgaris L. [Interestingly, quinoin was able to inhibit the virus infection when it was also applied on the abaxial leaf surface separately from the TNV that was inoculated on the adaxial surface. In this case, the reduction in the necrotic lesions with 2.0 µg/mL of quinoin was still very high (−94.72%) suggesting a synergistic action of this RIP against both viral and ribosomal RNA. Indeed, this evidence shows that there is no need for direct contact between virus and toxin; the virus inhibition would mainly occur inside the plant cells at the onset of infection . In thisgaris L. ,42.RIPs are a class of defence proteins that could act as antimicrobial agents. Indeed, their antipathogenic activities have been reported against both bacteria and fungi, which makes them active subjects in plant defence ,17,43.P. syringae pv. phaseolicola and P. syringae pv. actinidiae  seeds and exhibits toxicity against different cell lines in vitro. In this framework, this toxin represents a possible anti-nutritional factor in quinoa seeds besides the presence of saponins, phytic acid, tannins, oxalates and trypsin inhibitors, commonly known as anti-nutritional factors [Quinoin is a type 1 RIP found in  factors . Indeed, factors ,47. In tC. quinoa genome analysis and mass spectrometry. This toxin is a glycoprotein and its polypeptide chain consists of 254 amino acid residues, without cysteinyl residues. Similarities in the protein database have been found with other known RIPs and a predictive three-dimensional structure model obtained by homology modeling showed a typical RIP-fold [P. americana, and seems to be necessary in gene regulation during biotic and abiotic stresses [Thus, in this work, the amino acid sequence of quinoin was determined by using a combined approach based on RIP-fold . In addiRIP-fold . Throughstresses . MoreoveC. parasitica strains.Finally, the antipathogenic activities of quinoin against several types of pathogens  were evaluated. Quinoin possesses a strong antiviral activity towards TNV, even stronger than other previously tested RIPs. Interestingly, our data showed that this antiviral activity likely depends on both protein synthesis inhibition (N-glycosylase activity against ribosomes in cells) and depurination action on RNA virus genomes (activity verified in vitro on both TNV and TMV RNAs). In addition, this toxin showed a growth inhibitory effect on plant pathogen bacteria and a slight effect when tested against two Overall, this investigation improves the knowledge of the structural features and the different antipathogenic activities of quinoin. In particular, the antipathogenic activities of this toxin could be used for tuning its possible use as a biotechnological tool.Chemicals for protein purification chromatography were obtained as previously reported ,49. OtheQuinoin and minor forms of type 1 RIPs were purified according to the procedure published by Landi et al. (2021). In addition, for the purification of minority forms, after the last chromatography step on a CM-Sepharose column equilibrated in 5.0 mM Na/phosphate, pH 7.2 (buffer A) and eluted in the same buffer with a NaCl (buffer B) gradient up to 0.15 M , a further purification step was introduced. In particular, the minor forms were re-chromatographed by FPLC on an AKTA Purifier System , using a Source 15S PE 4.6/100 column, equilibrated in buffer A and eluted applying a linear gradient from 0 to 50% of buffer A containing NaCl 0.3 M over 60 min (flow rate 1.0 mL/min).The depurination assay (rRNA N-glycosylase assay) was conducted as previously described  on rabbiQuinoin homogeneity was determined by SDS-PAGE with a Mini-Protean II  using a 6% stacking and 15% separating polyacrylamide gel under reducing conditions; a precision plus protein kit (Bio-Rad) was used as reference proteins. Protein concentration was determined by a Pierce BCA Protein Assay Kit . Protein desalting was obtained by a reversed-phase HPLC (RP-HPLC) on a C-4 column  as previously reported . The pri4)HCO3 containing 10% acetonitrile. Subsequently, the protein was incubated with TPCK-treated trypsin, chymotrypsin or endoproteinase Arg-C with an enzyme/substrate 1:50 (w/w) final ratio added in three steps (the first at 0 h (E/S: 1:200); the second at 4 h (E/S: 1:100) and the third at 12 h ; 16 h total). Mixtures were then incubated at 37 °C, acidified (pH < 3.0) and frozen for subsequent analyses [C. quinoa seeds was carried out as reported above for quinoin.The chemical fragmentation of quinoin 50 µg) with cyanogen bromide (CNBr) was carried out in 70% TFA as a solvent as previously described [ µg with analyses . The TryMass spectrometry analyses on quinoin tryptic or chymotryptic digests (50 fmol) were performed on a Q-Exactive Orbitrap mass spectrometer equipped with an EASY-Spray nanoelectrospray ion source  and coupled to a Thermo Scientific Dionex UltiMate 3000RSLC nano-system . The solvent composition was 0.1% formic acid (FA) in water (solvent A) and 0.1% FA in acetonitrile (solvent B). Peptides were loaded on a trapping PepMapTM100 μCartridge column C-18  and desalted with solvent A for 3 min with at a flow rate of 10 μL/min. After trapping, eluted peptides were separated on an EASY-Spray analytical column . The run condition involved heating to 35 °C and a flow rate of 300 nL/min. The gradient for the peptides separation was 4% B for 3 min, from 4 to 55% B in 60 min, from 55 to 70% B in 10 min, and from 70 to 95% B in 2 min. Eluting peptides were analysed on the Q-Exactive mass spectrometer operating in positive polarity mode set-up as reported in previous works .r) of desalted quinoin together with the peptides from the enzymatic and chemical hydrolyses were determined by using a MALDI-TOF mass spectrometer  as previously reported [The relative molecular mass .The Glycomod tool was used to predict theoretical glycosidic chains  by using BLAST . Only reviewed sequences  were used for subsequent analyses. The signal peptide, connecting peptide and processed peptide sequences were removed in order to use only mature protein sequences. The sequence alignment was performed using the ClustalW tool available online . The similarity/identity matrix was obtained using the Sequence Identity and Similarity (SIAS) tool . The sequence logo was obtained using WebLogo tool available online . The evolutionary history was performed by using MEGA 10.0.5  [RIPs similar to quinoin were obtained from the Uniprot database (ne 2021) . For thihttps://zhanglab.ccmb.med.umich.edu/I-TASSER/ (visit date 8 June 2021). The selection of templates was performed by using the I-TASSER that at first identifies structure templates by LOMETS from the PDB library. The online procedure yielded the three-dimensional model on the basis of the multiple-threading alignments by LOMETS and the iterative TASSER simulations [A model of the three-dimensional structure of quinoin was obtained using the automated I-TASSER service available at the site ulations . The preulations .https://www.ncbi.nlm.nih.gov/genbank/; visit date 8 June 2021). The genomic sequence for NW_018745569.1 was obtained from C. quinoa cultivar QQ74.The study of the genomic sequence of the quinoin gene was performed by using the tools available on GenBank  was propagated in protocol . The Tobprotocol .P. vulgaris, cv. Borlotto Nano Lingua di Fuoco, were inoculated on the adaxial surface by rubbing them with a mixture of quinoin at different concentrations (10 to 0.2 µg/mL) and purified TNV (1:1), using a 600-mesh carborundum as an abrasive. The final virus concentration in the inoculum was 100 ng/mL. In some other experiments, quinoin was applied on the abaxial leaf surface while the virus was inoculated on the adaxial one in order to avoid contact between them. As controls, some plants were inoculated respectively with TNV alone, or with quinoin at different concentrations or water. Plants were observed for the development of lesions for up to 5 days. When the lesions were fully developed, the infected leaves were detached and immediately scanned at a 300 DPI resolution. Images were analysed with Global Lab  to determine the necrotic lesion area. The quinoin inhibitory activity was calculated as a percentage of the reduction in this area in respect to the leaves only inoculated with TNV. In total, 24 leaves from three independent experiments were analysed for each treatment.Fully expanded cotyledonary leaves of The N-glycosylase activity of RIPs was assayed in 100 μL samples containing 5.0 μg of TNV or TMV RNA, which were incubated with 3.0 μg of quinoin for 1 h at 37 °C. After treatment, the RNA was analysed by phenolization, treated with 1.0 M aniline acetate (pH 4.5), precipitated with ethanol and separated by gel electrophoresis (15 mA for 1 h) and visualised by ethidium bromide staining [Salmonella enterica subsp. enterica serovar enteridis ATCC 13,076 and Gram-positive Staphylococcus aureus, Pseudomonas syringae pv. phaseolicola and Pseudomonas syringae pv. actinidiae. A single colony of each strain was inoculated with 10 mL of Luria Broth  and incubated overnight at 37 and 30 °C for human and plant pathogens, respectively. A total of 100 µL of the bacterial culture was spread over LB agar plates. To check the antimicrobial activity of quinoin, 10 μL of the different diluted solution  were loaded on to 4 mm filter paper discs and placed on the inoculated plates. For reference, 10 µL of MilliQ water was added on the paper dish in the same inoculated plates. Plates were incubated for 18 h at 37 and 30 °C for human and plant pathogens, respectively. The inhibition zone was visualised by crystal violet staining as previously described [The agar diffusion assay was carried out using two different human pathogenic species of Gram-negative and positive bacterial strains and two Gram-plant pathogenic strains. Gram-negative escribed , with soCryphonectria parasitica strains (E4 and E13) deposited in the collection of the Agri-food and Environmental Microbiology Platform (PiMiAA), University of Brescia, Italy. The radial growth inhibition assay was carried out as previously described [The antifungal activity of quinoin was evaluated from two escribed . Brieflyescribed .


N-vinylcaprolactam) (PNVCL) polymers prepared by reversible addition–fragmentation chain transfer (RAFT) polymerization were subjected to aminolysis reaction using hexylamine. Chemically crosslinked gels or highly end-functionalized star polymers can be obtained depending mainly on the type of solvent used during the transformation of the RAFT functional group. An increase in the viscosity of the solution was observed when the aminolysis was carried out in THF. In contrast, when the reaction was conducted in dichloromethane, chain-end thiol (PNVCL)6 star polymers could be obtained. Moreover, when purified (PNVCL-SH)6 star polymers are in contact with THF, the gelation occurs in just a few minutes, with an obvious increase in viscosity, to form physical gels that become chemically crosslinked gels after 12 h. Interestingly, when purified (PNVCL-SH)6 star polymers were stirred in distilled water, even at high aqueous solution concentration (40 mg/mL), there was no increase in the viscosity or gelation, and no evident gels were observed. The analysis of the hydrodynamic diameter (hD) by dynamic light scattering (DLS) did not detect quantifiable change even after 4 days of stirring in water. On the other hand, the thiol groups in the (PNVCL-SH)6 star polymers were easily transformed into trithiocarbonate groups by addition of CS2 followed by benzyl bromide as demonstrated by UV-Vis spectroscopical analysis and GPC. After the modification, the (PNVCL)6 star polymers exhibit an intense yellow color typical of the absorption band of trithiocarbonate group at 308 nm. To further demonstrate the highly effective new trithiocarbonate end-functionality, the PNVCL polymers were successfully chain extended with N-isopropylacrylamide (NIPAM) to form six-arm star-shaped PNIPAM-b-PNVCL block copolymers. Moreover, the terminal thiol end-functionality in the (PNVCL-SH)6 star polymers was linked via disulfide bond formation to l-cysteine to further demonstrate its reactivity. Zeta potential analysis shows the pH-responsive behavior of these star polymers due to l-cysteine end-functionalization. By this using methodology and properly selecting the solvent, various environment-sensitive star polymers with different end-groups could be easily accessible.In this study, six-arm star-shaped poly( N,N-diethylacrylamide) RAFT-made polymers do not form disulfide groups easily, even though the purification process is long and involves several steps, such as dialysis against THF for 5 days. The SEC chromatogram of the thiolated polymer shows no evidence of macromolecular oxidation, although tris(2-carboxyethyl) phosphine hydrochloride (TCEP) was added after dialysis [N-isopropylacrylamide) prepared by RAFT polymerization in the presence of a catalytic amount of TCEP as a reducing agent. The aminolysis was performed in THF at room temperature for 1 h under N2 atmosphere. In the GPC traces, a small shoulder on the high molar mass side was observed, which is attributed to disulfide formation, in the presence or absence of TCEP. The authors consider that the oxidation of α,ω-thiol end polymers may occur during the purification of the polymer. The thiol functionality in the polymers was determined in 95% (Ellman test).Polymers prepared by reversible addition–fragmentation chain transfer (RAFT) polymerization can be transformed just by eliminating the RAFT group ,2,3,4,5.dialysis . In contdialysis  reportedAfter aminolysis of the RAFT-made polymers, several examples prove the successful reactivity of thiol chain-end, e.g., with isocyanates , with 2,To date, there is abundant literature related to the aminolysis of linear polymers prepared via RAFT ,7,8,9,10N-vinylcaprolactam) (PNVCL) is a thermosensitive, nonionic polymer that exhibits an LCST in water at around 32 °C. In the case of PNVCL, the transition temperature depends on the molecular weight, polymer concentration and presence of hydrophobic or polar groups at the chain-ends [Poly6 star polymers with thiol end functionality could be obtained. When these polymers were left to stir in distilled water at ambient temperature for several days, no gel formation was observed. Moreover, the transformation of the reactive thiol chain-end into a trithiocarbonate was also demonstrated. To the best of our knowledge, this transformation on a polymer has not yet been reported. In addition, to further demonstrate the chain-end functionality of the thiols in the (PNVCL-SH)6 star polymers, l-cysteine was coupled via disulfide bond formation.In this work, the xanthate end-groups in star-shaped PNVCL polymers were removed using hexylamine see . When th2SO4), potassium ethyl xanthogenate , N-isopropylacrylamide , benzyl bromide , hexylamine , L-cysteine  and NVCL  were purified by recrystallization with diethyl ether. Hydrochloric acid , ethanol , tetrahydrofuran , dimethylformamide , chloroform  and p-dioxane  were used as received. Column chromatographic purifications were performed using silica gel .2-Bromopropionyl bromide , dipentaerythritol, , triethylamine , 4,4-azobis (ACVA) , 2,2′-azobis , carbon disulfide , anhydrous sodium sulphate (NahD) and polydispersity index (PDI) were calculated using Malvern Instruments dispersion technology software, based on CONTIN analysis and Stokes–Einstein equation for spheres as usual. The LCST was taken as the temperature at which the star polymer was still soluble (just before the solution started to turn cloudy) at a 1 mg/mL concentration in water. The LCST was measured by DLS using the Nano-ZS Nanosizer with a temperature program that increased from 20 to 50 °C in two-degree steps, equilibrating for 4 min once the measurement temperature was achieved; measurements were performed three times, each of which included three 30 s runs. Gel permeation chromatography (GPC) was performed on a Varian 9002 chromatograph equipped with a series of three columns  and two detectors: a refractive index detector (Varian RI-4) and a triangle light scattering detector . The measurements were performed in THF at 35 °C. Polystyrene standards were used for calibration of the LS detector. THF was used as the mobile phase at a flow rate of 0.7 mL/min. Sample solutions were prepared using 20 mg/mL concentration and filtered through a 0.45 μm PTFE membrane filter before analysis.Dynamic light scattering (DLS) measurements were carried out using a Malvern Instruments Nano-ZS Nanosizer (ZEN 3690). The instrument is equipped with a helium–neon laser (633 nm) with a size detection range of 0.6 nm–5 μm. DLS experiments were performed at the scattering angle of 90° and equilibrated for 10 min before data collection. The solutions/dispersions were filtered through a 0.45 μm nylon membrane filter before analysis to remove dust. The volume-average hydrodynamic diameter (1H and 13C NMR spectra were collected on a Bruker AMX-400 (400 MHz) spectrometer and are reported in ppm using TMS as internal standard. The solvent used was deuterated chloroform (CDCl3) for all samples. UV-Vis spectra of the PNVCL polymers and copolymers were recorded using a UV-Vis Varian Cary 100 Spectrophotometer at room temperature.The synthesis of the linear and star PNVCL polymers was performed following the methodology reported previously ,19; see 48)6 star polymers  was added. The reaction mixture was stirred at room temperature for 10 min, then the polymer was precipitated by adding an excess of ethyl ether/petroleum ether (1:1), maintaining vigorous agitation. During the entire purification process, the vial must remain capped. The liquid phase was decanted, and the polymer product was redissolved in the minimum amount of dichloromethane and precipitated again with ethyl ether/petroleum ether (1:1); this procedure was repeated three times. In this step, the excess of hexylamine and the by-products were removed. The polymer product was dried under vacuum . These purification processes must be carried out within 15 min, and it is recommended to start the next transformation step as soon as possible, although the thiol-terminated polymer can be stored in a freezer without any change for up to two weeks. To analyze the sample on the GPC equipment, one purification is sufficient. The (PNVCL48-SH)6 star polymers were obtained as white solids (80%), nMGPC = 47,260 g/mol and Ð = 1.07 . A thick yellowish oil was obtained.The solvent waste from the first reprecipitation of the aminolysis reaction carried out with petroleum ether/ethyl ether was concentrated in vacuo. The product was purified using column chromatography on silica gel (CH32)6 was the precursor for this reaction  were dissolved in 5 mL of CHCl3. Then, triethylamine  was added, followed by the addition of CS2 . After stirring for 3 min, benzyl bromide  was added. The reaction was stirred for an additional 2 h at room temperature. The reaction product was precipitated by adding an excess of ethyl ether, maintaining vigorous agitation. The solvent was discarded, and the resulting polymer was redissolved in the minimum amount of dichloromethane and precipitated again with ethyl ether; this procedure was repeated three times. The polymer product was dried in vacuo. Then, the polymer was redissolved in THF, and the salt was removed by ultracentrifugation cycle . This purification method is more efficient than washing with dilute HCl and NaHCO3. The star polymer containing trithiocarbonate-type functional groups was obtained as a yellow solid (80%), .The sample (PNVCL55)6 6 trithiocarbonate-type polymers (macro-RAFT agent)  , NIPAM  and ACVA  were dissolved in 3 mL of DMF. The NIPAM/macro-RAFT agent/ACVA ratio was 200/5/1. The solution was deoxygenated by bubbling nitrogen for 20 min at room temperature. Then, the flask was placed in an oil bath preheated at 70 °C. After 12 h, the polymerization was stopped by cooling to room temperature. Then, the polymer was precipitated by adding an excess of ethyl ether, maintaining vigorous agitation. The resulting polymer was dried under vacuum and redissolved in the minimum amount of dichloromethane and precipitated again with ethyl ether; this procedure was repeated three times. Finally, the copolymer product was dried under vacuum. The polymer, star (PNVCL55-b-PNIPAM19%)6 , was obtained as a yellowish solid (35%).The starting xanthate polymer for this reaction was . After 5 min, 3 mL of ethanol was added. The reaction mixture was stirred for 72 h. The remaining l-cysteine was removed by ultracentrifugation . The solvent was removed under reduced pressure. The polymer was redissolved in dichloromethane, and an ultracentrifugation cycle was carried out to remove traces of l-cysteine. This last step is important to gain the total solubility of the polymer product in THF before its analysis in GPC. The resulting polymer was dried under vacuum. (PNVCL-S-S-cysteine)6 star polymers  were obtained with 95% yield.In a 20 mL vial containing a magnetic stir bar, 0.5 g of star (PNVCL-SH)2) [The generation of reactive terminal thiol polymers through an aminolysis reaction with the aim of postfunctionalization is quite relevant for precision polymer synthesis. To achieve this, it is necessary to avoid the formation of disulfide bonds between the polymer chain ends. Some of the influencing factors could be the chemical structure of the polymer, the amine type, the solvent, the reaction time and the atmosphere  ,2,3,4,5.6 star polymers. The xanthate group was removed using hexylamine (8-fold in excess for each arm) in THF solution, at ambient conditions. It is important to mention that the concentration of the polymer in the solution was 37 mg/mL.According to a review of the literature, the removal of the thiocarbonylthio group is mostly carried out in THF as solvent. Therefore, based on these facts, this study began seeking the aminolysis of the xanthate group in the RAFT-made (PNVCL)6 star product and the starting (PNVCL-xanthate)6 star polymers are shown. The thiol-containing samples taken out at 5, 10 and 15 min of reaction show a prominent shoulder on the high-molar-mass side of the chromatogram which can be attributed to the formation of disulfide groups between polymer chains. Moreover, the intensity of the main peak decreases as the reaction time increases. It is important to note that the main peak for the (PNVCL-SH)6 star polymers is shifted to larger retention times  into a sulfine group (O=C–S–) ,6.Although the removal of the RAFT groups through aminolysis in a DMF solution has been reported in the literature, it is not recommended here, because the polymer purification process after aminolysis is complicated due to the inherent polarity of DMF, delaying the polymer purification. Dichloromethane was the selected solvent for the aminolysis reaction of the polymers. The reaction time was set at only 10 min because at 5 min there is no longer an absorbance of the thiocarbonylthio group related to the xanthate according to the UV-Vis analysis, as will be shown later.Based on the literature, the reaction for the removal of the RAFT group by aminolysis can be carried out in times ranging from seconds to several hours ,21,22,23c = 20 mg/mL), the formation of disulfide groups seems to start rapidly. After purification, the sample was dissolved in THF and stirred only for 30 s; then, it was passed through the filter and quickly injected into the GPC equipment.The GPC analysis procedure for thiol-functionalized star polymers also had to be optimized since as soon as the samples are exposed to THF hane see b were alvely see b. On theO-ethyl hexylcarbamothioate  was observed . Solutions of linear polymers in THF were prepared and left stirring for different periods of time  to later be analyzed by GPC. In rved see . This innMGPC = 31,000 g/mol, Ð = 1.35) exhibits practically twice the molecular weight with respect to the starting PNVCL polymer.The sample analyzed at 5 h was bimodal with a shoulder at lower retention time. The associate peak has almost the same intensity that the main peak. For the sample at 10 and 24 h, it is observed that the intensity of the main peak decreases while the associated peak increases. Note that the chromatogram for the sample at 24 h 6 star polymers after stirring 10 min in THF. Regardless of polymer concentration, the hD was around 8–10 nm. The samples were kept stirring in THF, and the hD was analyzed after 2 and 72 h. In the case of the star-shaped (PNVCL-SH)6 polymers, the sample at 1 mg/mL exhibits hD values of 32 nm after 2 h and 59 nm after 72 h of stirring 6 star polymers stirring a. The nion see . The polIn contrast, benzyl bromide reacts with the trithiocarbonate anion very efficiently, resulting in a yellow polymer after the purification step. The xanthate moiety and the trithiocarbonate moiety have different UV absorptions from each other. In 6 star polymers with xanthate, thiol and trithiocarbonate groups are shown. The chromatogram of the thiol end sample  is shifted at a longer retention time and slightly higher molecular weight, but after the postfunctionalization, the position of the peak of the trithiocarbonate polymer  was practically the same as that of the starting xanthate polymer 6 star polymer is shown in The N-vinylpyrrolidone)-based block copolymers by RAFT polymerization under irradiation of UV or visible light. The PMAMs  were used as the macro-RAFT agents. In addition, the living character of polymerization was demonstrated.The preparation of block copolymers involving “less activated” monomers (LAM) and “more activated” monomers (MAM) by controlled radical polymerization is still a challenge ,28,29. T6 star polymers transformed from xanthate into trithiocarbonate-functional groups was demonstrated with the polymerization of N-isopropylacrylamide. The polymerization was performed in DMF at 70 °C using ACVA as an initiator.The living character of the (PNVCL)1H NMR spectrum displayed in H–) of the PNVCL block is observed at 4.39 ppm “b”. For the PNIPAM block, the hydrogen of the amide group (–NH–) “d” was observed at 7.04–6.0 ppm, and the methine (–NHCH–) “c” was observed at 4.0 ppm. The molar composition of each block was determined by comparing the integration value of the methine peak “b” of the PNVCL block at 4.49 ppm and the methine peak for PNIPAM at 4.0 ppm “c”. This calculation demonstrated that the copolymer has a PNIPAM content of 19%.The With this simple and fast transformation of xanthates to trithiocarbonates presented here, the possibility of generating a great variety of block copolymers is opened by combining LAM monomers such as NVCL and MAM monomers such as methacrylates.6 star polymers  and the (PNVCL-b-PNIPAM)6 star block copolymers  are shown. As can be seen, the GPC chromatogram for the star block copolymer was shifted to shorter retention times compared to the starting star polymer.In 55)6 star polymers and (PNVCL55-b-PNIPAM19%)6 star block copolymers was evaluated for both heating and cooling cycles. 55)6 star polymers. PNVCL does not show hysteresis normally [55-b-PNIPAM50%)6 star block copolymer is remarkable for the cooling process  by stirring in ethanol or DMF for 72 h.The thiol end-groups in the star-shaped (PNVCL-SH)6 functionalized star polymer was dissolved in THF (20 mg/ mL) and was left under observation for 24 h to be analyzed later by GPC. We mentioned above that the thiol-terminated polymers dissolved in THF tend to form gels quickly. So, the fact that the sample could be analyzed without problems by GPC proves the transformation of the thiol group.The sample of (PNVCL-S-S-cysteine)6 star polymer was apparently increased  with respect to the starting xanthate polymer 6 and the thiol end polymer were also studied . As the pH increased, the value of the zeta potential became negative. For example, at a pH value of 8.57, the zeta potential value was strongly negative: −29.9 mV. The pH-responsive behavior of the (PNVCL-S-S-cysteine)6 was higher compared with the thiolated and the (PNVCL35-xanthate)6 star polymer. In the thiol end sample , the zeta potential value was −9.81 mV 6 star polymer dissolved in distilled water presented a pH value of 7.01 and a zeta potential value of −10.2 mV , and itsted see c. Moreov2 mV see . PNVCL p2 mV see . In any N-vinylcaprolactam) polymers were prepared by aminolysis reaction using hexylamine in dichloromethane as solvent. (PNVCL-SH)6 polymers are precursors of either cross-linked gels or highly effective postfunctionalized star polymers, depending on the selected solvent. When the thiol end-capped star polymers are in contact with THF, they form gels rapidly. In contrast, (PNVCL-SH)6 polymers dissolved and stirred for days in distilled water do not form gels. The thiol groups in the (PNVCL-SH)6 star polymers were transformed into trithiocarbonate RAFT groups. After the modification, the star polymers exhibit an intense yellow color typical of the absorption band of the trithiocarbonate group at 308 nm. The (PNVCL-trithiocarbonate)6 star polymers were successfully chain extended with N-isopropylacrylamide (NIPAM) to form six-arm star-shaped PNIPAM-b-PNVCL block copolymers. Moreover, the terminal thiol end-functionality in the (PNVCL-SH)6 star polymers was linked via disulfide bond formation to L-cysteine to further demonstrate its reactivity. Zeta potential analysis shows the pH-responsive behavior of these star polymers due to the L-cysteine end-functionalization. The LCST values of the PNVCL star polymers change as expected with molecular weight, end-group functionalization and comonomer.Thiol-terminated star-shaped poly(